WO2010131452A1 - Ttk peptides and vaccines including the same - Google Patents

Ttk peptides and vaccines including the same Download PDF

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Publication number
WO2010131452A1
WO2010131452A1 PCT/JP2010/003166 JP2010003166W WO2010131452A1 WO 2010131452 A1 WO2010131452 A1 WO 2010131452A1 JP 2010003166 W JP2010003166 W JP 2010003166W WO 2010131452 A1 WO2010131452 A1 WO 2010131452A1
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Prior art keywords
peptide
present
cancer
peptides
ttk
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PCT/JP2010/003166
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English (en)
French (fr)
Inventor
Yusuke Nakamura
Takuya Tsunoda
Ryuji Ohsawa
Sachiko Yoshimura
Tomohisa Watanabe
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Oncotherapy Science, Inc.
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Priority to CN201080031259.2A priority Critical patent/CN102459314B/zh
Priority to SG2011082963A priority patent/SG175998A1/en
Priority to US13/320,022 priority patent/US8530430B2/en
Priority to RU2011150283/04A priority patent/RU2531348C2/ru
Priority to CA2761393A priority patent/CA2761393A1/en
Priority to MX2011012013A priority patent/MX2011012013A/es
Application filed by Oncotherapy Science, Inc. filed Critical Oncotherapy Science, Inc.
Priority to EP10774711A priority patent/EP2430039A4/en
Priority to JP2011548451A priority patent/JP5786178B2/ja
Priority to BRPI1013938A priority patent/BRPI1013938A2/pt
Priority to AU2010248702A priority patent/AU2010248702A1/en
Publication of WO2010131452A1 publication Critical patent/WO2010131452A1/en
Priority to IL215915A priority patent/IL215915A0/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the field of biological science, more specifically to the field of cancer therapy.
  • the present invention relates to novel peptides that are extremely effective as cancer vaccines and drugs for treating and preventing tumors.
  • CD8 positive CTLs recognize epitope peptides derived from the tumor-associated antigens (TAAs) found on the major histocompatibility complex (MHC) class I molecule, and then kill the tumor cells.
  • TAAs tumor-associated antigens
  • MHC major histocompatibility complex
  • TAAs small cell lung cancers
  • PLCs small cell lung cancers
  • PTL2/WO2007/013671 esophageal cancers
  • preferred TAAs should be expressed primarily by cancer cells, with limited or no expression by normal healthy tissues.
  • Preferred TAAs as immunotherapy targets are those that are indispensable for proliferation and survival of cancer cells. Such TAAs may minimize the well-described risk of immune escape of cancer cells attributable to deletion, mutation, or down-regulation of TAAs as a consequence of therapeutically driven immune selection.
  • TTK Protein kinase was identified as one of the genes up-regulated in lung cancer (NPL3/Kikuchi at al., Int J Oncol. 2006 Apr;28(4):799-805). Expression of TTK is specifically up-regulated in tumor cells in more than 80% of the patients with lung cancer and esophageal cancer. At the same time, TTK is not expressed in any other normal vital organ, except the testis. Taken together, these facts suggest TTK may be applicable as a target of cancer immunotherapy for patient with TTK up-regulated tumors.
  • TTK3 Peptides derived from TTK that have specific CTL inducibility against target cells exogenously expressing TTK and HLA-A*0201 have previously been disclosed (See WO2008/102557 (PTL3), the results of which are duplicated herein as Figure 4).
  • the present invention is based, at least in part, on the discovery of novel peptides that may serve as targets of immunotherapy. Because TAAs are sometimes perceived by the immune system as "self” and therefore often have no innate immunogenicity, the discovery of appropriate targets is of extreme importance.
  • TTK a typical amino acid sequence and gene sequence are shown in SEQ ID NO: 40 and SEQ ID NO: 39, respectively, but are not limited to, and a typical gene sequence is also available from, for example, GenBank Accession No.
  • NM_003318 has been identified as up-regulated in cancers, including, but not limited to, lung cancer, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, Chronic myelogenous leukemia (CML), colorectal cancer, endometriosis, esophageal cancer, gastric cancer, diffused type gastric cancer, liver cancer, non-small cell lung cancer (NSCLC), lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma, small cell lung cancer (SCLC), soft tissue tumor and testicular tumor.
  • TTK tumor necrosis
  • SCLC small cell lung cancer
  • the present invention further relates to the identification of specific epitope peptides of the gene products of TTK that possess the ability to induce CTLs specific to TTK.
  • peripheral blood mononuclear cells PBMCs
  • HLA-A*0201 binding candidate peptides derived from TTK were stimulated using HLA-A*0201 binding candidate peptides derived from TTK.
  • CTL lines were then established with specific cytotoxicity against the HLA-A2 positive target cells pulsed with each of candidate peptides.
  • CTL lines induced with a peptide having an amino acid sequence of SEQ ID NO: 3 showed significantly potent specific cytotoxicity against cells expressing HLA-A*0201 and TTK.
  • peptides that bind to HLA antigen, particularly those that include an amino acid sequence of TTK (e.g., SEQ ID NO: 40) or an immunologically active fragment thereof.
  • TTK e.g., SEQ ID NO: 40
  • These peptides are expected to have CTL inducibility and, thus, can be used to induce CTL ex vivo or to be administered to a subject for inducing immune responses against cancers such as lung cancer, bladder cancer, breast cancer, cervical cancer, cholangincellular carcinoma, CML, colorectal cancer, endometriosis, esophageal cancer, gastric cancer, diffused type gastric cancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma, SCLC, soft tissue tumor and testicular tumor.
  • Preferred peptides are peptides having an amino acid sequence of SEQ ID NO: 3.
  • the peptides of the present invention encompass those wherein one, two or more amino acids are substituted, deleted and/or added, so long as the resulting modified peptides retain the original CTL inducibility.
  • the present invention also provides isolated polynucleotides encoding any peptides of the present invention. These polynucleotides can be used for inducing or preparing APCs with CTL inducibility or can be administered to a subject for inducing immune responses against cancers much like the present peptides.
  • one object of the present invention is to provide agents or compositions that induce CTLs, such agents or compositions including one or more peptides of the present invention or polynucleotides encoding such peptides.
  • the present invention further contemplates pharmaceutical agents or compositions including one or more peptides of the present invention or polynucleotides encoding such peptides, such agents or compositions formulated for the treatment and/or prophylaxis of cancers, as well as the prevention of postoperative recurrence thereof, such cancers including, but not limited, lung cancer, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophageal cancer, gastric cancer, diffused type gastric cancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma, SCLC, soft tissue tumor and testicular tumor.
  • the present pharmaceutical agents or compositions can include APCs or exosomes that present any of the present peptides instead of/in addition to the present peptides or polynucleotides as active ingredients.
  • the peptides and polynucleotides of the present invention can induce APCs that present on their surface a complex of an HLA antigen and the present peptide, for example, by contacting APCs derived from a subject with the peptide or introducing a polynucleotide encoding a peptide of the present invention into APCs.
  • APCs have high CTL inducibility against target peptides and thus find use in cancer immunotherapy. Accordingly, the present invention contemplates both methods for inducing APCs with CTL inducibility and APCs obtained by such methods.
  • the present invention also provides methods for inducing CTL, methods that include the step of co-culturing CD8 positive cells with APCs or exosomes presenting one or more peptides of the present invention on its surface or the step of introducing a gene that includes a polynucleotide encoding a T cell receptor (TCR) subunit polypeptide capable of bind to the present peptide.
  • TCR T cell receptor
  • CTLs obtained by such methods find use in the treatment and prevention of cancers, examples of which include, but are not limited to, lung cancer, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophageal cancer, gastric cancer, diffused type gastric cancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma, SCLC, soft tissue tumor and testicular tumor. Therefore, the present invention encompasses the CTLs obtained by the present methods.
  • the applicability of the present invention extends to any of a number of diseases relating to or arising from TTK overexpression, such as cancer, exemplary cancers including, but not limited to, lung cancer, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophageal cancer, gastric cancer, diffused type gastric cancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma, SCLC, soft tissue tumor and testicular tumor.
  • cancer exemplary cancers including, but not limited to, lung cancer, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophageal cancer, gastric cancer, diffused type gastric cancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma, SCLC, soft tissue tumor and
  • the present invention provides the following [1] to [21]; [1] An isolated peptide comprising an amino acid sequence of SEQ ID NO: 3. [2] An isolated peptide, having cytotoxic T lymphocyte (CTL) inducibility, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 3, wherein 1, 2, or several amino acid(s) are substituted, deleted, or added, [3] The isolated peptide of [2], wherein the peptide has one or both of the following characteristics: (a) second amino acid from the N-terminus is or is modified to be an amino acid selected from the group consisting of leucine and methionine; and (b) C-terminal amino acid is or is modified to be an amino acid selected from the group consisting of valine and leucine, [4] The isolated peptide of any one of [1] to [3], wherein said peptide is nonapeptide, [5] An isolated polynucleotide encoding the peptide of any one of [1] to [1
  • Figure 1 is composed of a series of photographs, (a) to (j), depicting the results of IFN-gamma ELISPOT assays on CTLs that were induced with peptides derived from TTK.
  • the CTLs in the following well numbers showed potent IFN-gamma production as compared with the control: well #1 and #4 stimulated with TTK-A02-9-462 (SEQ ID NO: 1) (a), #7 with TTK-A02-9-630 (SEQ ID NO: 2) (b), #5 with TTK-A02-9-593 (SEQ ID NO: 3) (c), #4, #5, #6 and #7 with TTK-A02-9-719 (SEQ ID NO: 6) (d), #5 and #7 with TTK-A02-9-142 (SEQ ID NO: 15) (e), #7 with TTK-A02-9-146 (SEQ ID NO: 19) (f), #5, #7 and #8 with TTK-A02-9-564 (SEQ ID NO: 20) (g),
  • Figure 2a-d is composed of a series of line graphs, (a) to (d), depicting the results of an IFN-gamma ELISA assay demonstrating the IFN-gamma production of CTL lines stimulated with TTK-A02-9-462 (SEQ ID NO: 1) (a), TTK-A02-9-630 (SEQ ID NO: 2) (b), TTK-A02-9-593 (SEQ ID NO: 3) (c), and TTK-A02-9-719 (SEQ ID NO: 6) (d).
  • the results demonstrate that CTL lines established by stimulation with certain TTK peptides and the CTL clone established from the CTL line showed potent IFN-gamma production as compared with the control.
  • “+” indicates the IFN-gamma production against target cells pulsed with the appropriate peptides and "-" indicates the IFN-gamma production against target cells not pulsed with any peptides.
  • Figure 2e-g is composed of a series of line graphs, (e) to (g), depicting the results of an IFN-gamma ELISA assay demonstrating the IFN-gamma production of CTL lines stimulated with TTK-A02-9-142 (SEQ ID NO: 15) (e) and TTK-A02-10-462 (SEQ ID NO: 22) (f), and the IFN-gamma production of a CTL clone established from a CTL line stimulated with TTK-A02-9-593 (SEQ ID NO: 3) by the stimulation with the same peptide (g).
  • Figure 3 is composed of a series of line graphs, (a) to (c), depicting the specific CTL activity against the target cells that exogenously express TTK and HLA-A*0201 and tumor cells that are HLA-A2 positive and overexpress TTK.
  • COS7 cells transfected with HLA-A*0201 and the full length of TTK gene were prepared as target cells, while COS7 cells transfected with HLA-A*0201 or with the full length of TTK gene were prepared as controls, (a) and (b).
  • H1650 a tumor cell line which is HLA-A2 positive and overexpress TTK
  • PC-3 and TE-1 tumor cell lines which are HLA-A2 negative and overexpress TTK
  • no significant specific CTL activity was detected against target cells expressing either HLA-A*0201 (triangle) or TTK (circle).
  • the CTL clone also showed specific CTL activity against tumor cell line (HLA-A2 + , TTK + ): H1650 cells (c) (black box). On the other hand, no significant specific CTL activity was detected against target cells (HLA-A2 - , TTK + ) (triangle: PC-3, circle: TE-1).
  • Figure 4 is composed of a set of line graphs depicting the specific CTL inducing activity of peptides derived from TTK against target cells exogenously expressing TTK and HLA-A*0201 (Endo assay results).
  • the graphs corresponds to Figure 8b, c, d and e set forth in Applicants' WO2008/102557.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that similarly function to the naturally occurring amino acids.
  • Amino acid may be either L-amino acids or D-amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those modified after translation in cells (e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine).
  • amino acid analog refers to compounds that have the same basic chemical structure (an alpha carbon bound to a hydrogen, a carboxy group, an amino group, and an R group) as a naturally occurring amino acid but have one or more modified R group(s) or modified backbones (e.g., homoserine, norleucine, methionine, sulfoxide, methionine methyl sulfonium).
  • modified R group(s) or modified backbones e.g., homoserine, norleucine, methionine, sulfoxide, methionine methyl sulfonium.
  • amino acid mimetic refers to chemical compounds that have different structures but similar functions to general amino acids.
  • Amino acids may be referred to herein by their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • the terms "gene”, “polynucleotides”, “nucleotides” and “nucleic acids” are used interchangeably herein and, unless otherwise specifically indicated are, similarly to amino acids, referred to by their commonly accepted single-letter codes.
  • composition as used herein is intended to encompass a product including the specified ingredients in the specified amounts, as well as any product that results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
  • pharmaceutical composition is intended to encompass a product including the active ingredient(s), and any inert ingredient(s) that make up the carrier, as well as any product that results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients.
  • the phrase “pharmaceutical composition” encompasses any composition made by admixing a compound of the present invention and a pharmaceutically or physiologically acceptable carrier.
  • pharmaceutically acceptable carrier or “physiologically acceptable carrier”, as used herein, means a pharmaceutically or physiologically acceptable material, composition, substance or vehicle, including but not limited to, a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the active ingredient(s) from one organ, or portion of the body, to another organ, or portion of the body.
  • cancer refers to the cancers or tumors that overexpress TTK gene, examples of which include, but are not limited to, lung cancer, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophageal cancer, gastric cancer, diffused type gastric cancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma, SCLC, soft tissue tumor and testicular tumor.
  • cytotoxic T lymphocyte cytotoxic T cell
  • CTL cytotoxic T lymphocyte
  • cytotoxic T cell cytotoxic T cell
  • HLA-A2 representatively refers to the subtypes such as HLA-A*0201 and HLA-A*0206.
  • kit is used in reference to a combination of reagents and other materials. It is contemplated herein that the kit may include microarray, chip, marker, and so on. It is not intended that the term “kit” be limited to a particular combination of reagents and/or materials. To the extent that the methods and compositions of the present invention find utility in the context of the "treatment” of cancer, a treatment is deemed “efficacious” if it leads to clinical benefit such as, reduction in expression of TTK gene, or a decrease in size, prevalence, or metastatic potential of the cancer in the subject.
  • efficacious means that it retards or prevents cancers from forming or prevents or alleviates a clinical symptom of cancer. Efficaciousness is determined in association with any known method for diagnosing or treating the particular tumor type.
  • prevention and prophylaxis can occur “at primary, secondary and tertiary prevention levels.” While primary prevention and prophylaxis avoid the development of a disease, secondary and tertiary levels of prevention and prophylaxis encompass activities aimed at the prevention and prophylaxis of the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications. Alternatively, prevention and prophylaxis can include a wide range of prophylactic therapies aimed at alleviating the severity of the particular disorder, e.g. reducing the proliferation and metastasis of tumors.
  • the treatment and/or prophylaxis of cancer and/or the prevention of postoperative recurrence thereof include any of the following steps, such as the surgical removal of cancer cells, the inhibition of the growth of cancerous cells, the involution or regression of a tumor, the induction of remission and suppression of occurrence of cancer, the tumor regression, and the reduction or inhibition of metastasis.
  • Effective treatment and/or the prophylaxis of cancer decreases mortality and improves the prognosis of individuals having cancer, decreases the levels of tumor markers in the blood, and alleviates detectable symptoms accompanying cancer.
  • reduction or improvement of symptoms constitutes effectively treating and/or the prophylaxis include 10%, 20%, 30% or more reduction, or stable disease.
  • an antibody refers to immunoglobulins and fragments thereof that are specifically reactive to a designated protein or peptide thereof.
  • An antibody can include human antibodies, primatized antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, antibodies fused to other proteins or radiolabels, and antibody fragments.
  • an antibody herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
  • An “antibody” indicates all classes (e.g., IgA, IgD, IgE, IgG and IgM).
  • peptides derived from TTK function as an antigen recognized by CTLs
  • SEQ ID NO: 40 peptides derived from TTK (SEQ ID NO: 40) were analyzed to determine whether they were antigen epitopes restricted by HLA-A2 which are commonly encountered HLA alleles (Date Y et al., Tissue Antigens 47: 93-101, 1996; Kondo A et al., J Immunol 155: 4307-12, 1995; Kubo RT et al., J Immunol 152: 3913-24, 1994).
  • TTK-A02-9-462 SEQ ID NO: 1
  • TTK-A02-9-630 SEQ ID NO: 2
  • TTK-A02-9-593 SEQ ID NO: 3
  • TTK-A02-9-719 SEQ ID NO: 6
  • TTK-A02-9-142 SEQ ID NO: 15
  • TTK-A02-10-462 SEQ ID NO: 22
  • TTK is an antigen recognized by CTLs and that the peptides tested are epitope peptides of TTK restricted by HLA-A2.
  • CTLs established with TTK-A02-9-593 (SEQ ID NO: 3) showed more potent specific CTL activity against target cells expressing HLA-A*0201 and TTK than reported before.
  • TTK-A02-9-593 (SEQ ID NO: 3) is a suitable peptide to induce potent specific CTL activity against cells over-expressing TTK.
  • TTK gene is over-expressed in cancer cells and tissues, including, but not limited to, lung cancer, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophageal cancer, gastric cancer, diffused type gastric cancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma, SCLC, soft tissue tumor and testicular tumor but not expressed in most normal organs, it represents a good target for cancer immunotherapy.
  • the present invention provides nonapeptides (peptides consisting of nine amino acid residues) and decapeptides (peptides consisting of ten amino acid residues) corresponding to CTL-recognized epitopes from TTK.
  • the present invention provides isolated peptides that bind to HLA antigens and induce cytotoxic T lymphocytes (CTLs), wherein the peptide consists of the amino acid sequence of SEQ ID NO: 40 or is an immunologically active fragment thereof.
  • CTLs cytotoxic T lymphocytes
  • Particularly preferred examples of nonapeptides and decapeptides of the present invention include peptides having the amino acid sequence of SEQ ID NO: 3.
  • the present invention encompasses peptides composed of any fragments derived from TTK that bind with HLA antigens by such known programs. Furthermore, such peptides may include the peptide consisting of the full length of TTK.
  • the nonapeptides and decapeptides of the present invention may be flanked with additional amino acid residues, so long as the resulting peptides retain their CTL inducibility.
  • the additional amino acid residues may be composed of any kind of amino acids so long as they do not impair the CTL inducibility of the original peptide.
  • the present invention encompasses peptides with binding affinity to HLA antigens, including peptides derived from TTK.
  • Such peptides are, for example, less than about 40 amino acids, often less than about 20 amino acids, and usually less than about 15 amino acids.
  • modified peptides i.e., peptides composed of an amino acid sequence, in which one, two or several amino acid residues have been modified (i.e., substituted, added, deleted or inserted) as compared to an original reference sequence
  • modified peptides have been known to retain the biological activity of the original peptide (Mark et al., Proc Natl Acad Sci USA 1984, 81: 5662-6; Zoller and Smith, Nucleic Acids Res 1982, 10: 6487-500; Dalbadie-McFarland et al., Proc Natl Acad Sci USA 1982, 79: 6409-13).
  • the peptides of the present invention have both CTL inducibility and an amino acid sequence of SEQ ID NO: 3, wherein one, two or even more amino acids are added, deleted and/or
  • amino acid side chain characteristics that are desirable to conservative include, for example: hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (D, N, E, Q); a base containing side-chain (R, K, H); and an aromatic group containing side-chain (H, F, Y, W).
  • hydrophobic amino acids A, I, L, M, F, P, W, Y, V
  • hydrophilic amino acids R, D, N, C, E, Q, G, H, K, S, T
  • the following eight groups each contain amino acids that are accepted in the art as conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Aspargine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins 1984).
  • Such conservatively modified peptides are also considered to be peptides of the present invention.
  • peptides of the present invention are not restricted thereto and may include non-conservative modifications, so long as the resulting modified peptide retains the CTL inducibility of the original peptide.
  • modified peptides should not exclude CTL inducible peptides of polymorphic variants, interspecies homologues, and alleles of TTK.
  • modify insert, add, delete and/or substitute
  • a small number for example, 1, 2 or several
  • the term "several" means 5 or fewer amino acids, for example, 4 or 3 or fewer.
  • the percentage of amino acids to be modified is preferably 20% or less, more preferably 15% or less, and even more preferably 10% or less or 1 to 5%.
  • the present peptides When used in the context of cancer immunotherapy, the present peptides should be presented on the surface of a cell or exosome, preferably as a complex with an HLA antigen. Therefore, it is preferable to select peptides that not only induce CTLs but also possess high binding affinity to the HLA antigen. To that end, the peptides can be modified by substitution, insertion, and/or addition of the amino acid residues to yield a modified peptide having improved binding affinity.
  • peptides having an amino acid sequence of SEQ ID NO: 3 wherein the second amino acid from the N-terminus is substituted with leucine or methionine, and/or wherein the C-terminus is substituted with valine or leucine are encompassed by the present invention. Substitutions may be introduced not only at the terminal amino acids but also at the position of potential T cell receptor (TCR) recognition of peptides.
  • TCR T cell receptor
  • a peptide with amino acid substitutions may have equal to or better function than that of the original, for example, CAP1, p53 (264-272), Her-2/neu (369-377) or gp100 (209-217) (Zaremba et al. Cancer Res. 57, 4570-4577, 1997, T. K. Hoffmann et al. J Immunol. (2002) Feb 1;168(3):1338-47., S. O. Dionne et al. Cancer Immunol immunother. (2003) 52: 199-206 and S. O. Dionne et al. Cancer Immunology, Immunotherapy (2004) 53, 307-314).
  • the present invention also contemplates the addition of one, two or several amino acids may also be added to the N and/or C-terminus of the present peptides.
  • modified peptides having high HLA antigen binding affinity and retained CTL inducibility are also included in the present invention.
  • side effects such as autoimmune disorders or allergic symptoms against specific substances may be induced. Therefore, it is preferable to perform homology searches using available databases to avoid situations in which the sequence of the peptide matches the amino acid sequence of another protein.
  • the objective peptide may be modified in order to increase its binding affinity with HLA antigens, and/or increase its CTL inducibility without any danger of such side effects.
  • CTL inducibility indicates the ability of the peptide to induce cytotoxic T lymphocytes (CTLs) when presented on antigen-presenting cells (APCs).
  • CTL inducibility includes the ability of the peptide to induce CTL activation, CTL proliferation, promote CTL lysis of target cells, and to increase CTL IFN-gamma production.
  • Confirmation of CTL inducibility is accomplished by inducing APCs carrying human MHC antigens (for example, B-lymphocytes, macrophages, and dendritic cells (DCs)), or more specifically DCs derived from human peripheral blood mononuclear leukocytes, and after stimulation with the peptides, mixing with CD8 positive cells, and then measuring the IFN-gamma produced and released by CTL against the target cells.
  • human MHC antigens for example, B-lymphocytes, macrophages, and dendritic cells (DCs)
  • DCs dendritic cells
  • transgenic animals that have been produced to express a human HLA antigen (for example, those described in BenMohamed L, Krishnan R, Longmate J, Auge C, Low L, Primus J, Diamond DJ, Hum Immunol 2000 Aug, 61(8): 764-79, Related Articles, Books, Linkout Induction of CTL response by a minimal epitope vaccine in HLA A*0201/DR1 transgenic mice: dependent on MHC (HLA) class II restricted T(H) response) can be used.
  • the target cells may be radiolabeled with 51 Cr and such, and cytotoxic activity may be calculated from radioactivity released from the target cells.
  • CTL inducibility can be assessed by measuring IFN-gamma produced and released by CTL in the presence of APCs that carry immobilized peptides, and visualizing the inhibition zone on the media using anti-IFN-gamma monoclonal antibodies.
  • a nonapeptide consisting of the amino acid sequence indicated by SEQ ID NO: 3 showed particularly high CTL inducibility as well as high binding affinity to an HLA antigen.
  • such peptide is a preferred exemplified embodiment of the present invention.
  • the result of homology analysis showed that the peptide does not have significant homology with peptides derived from any other known human gene products. Accordingly, the possibility of unknown or undesired immune responses arising when used for immunotherapy is lowered. Therefore, also from this aspect, the peptide having an amino acid sequence of SEQ ID NO: 3 find use for eliciting immunity in cancer patients against TTK.
  • the peptides of the present invention preferably, peptides having an amino acid sequence of SEQ ID NO: 3.
  • the peptides of the present invention may also be linked to other peptides, so long as the resulting linked peptide retains the requisite CTL inducibility of the original peptide.
  • suitable other peptides include: other CTL inducible peptides derived from TTK (e.g., peptides having the amino acid sequence selected from among SEQ ID NO: 1, 2, 6, 15 and 22) or the CTL inducible peptides derived from other TAAs.
  • Suitable inter-peptide linkers are well known in the art, for example, AAY (P. M. Daftarian et al., J Trans Med 2007, 5:26), AAA, NKRK (R. P. M.
  • non-TTK tumor associated antigen peptides also can be used substantially simultaneously to increase the immune response via HLA class I and/or class II. It is well established that cancer cells can express more than one tumor associated gene. Thus, it is within the scope of routine experimentation for one of ordinary skill in the art to determine whether a particular subject expresses additional tumor associated genes, and then to include HLA class I and/or HLA class II binding peptides derived from expression products of such genes in TTK compositions or vaccines according to the present invention.
  • HLA class I and HLA class II binding peptides are known to one of ordinary skill in the art (for example, see Coulie, Stem Cells 13:393-403, 1995), and can be used in the present invention in a like manner as those disclosed herein.
  • those of ordinary skill in the art can readily prepare polypeptides including one or more TTK peptides and one or more of the non-TTK peptides, or nucleic acids encoding such polypeptides, using standard procedures of molecular biology.
  • polytopes i.e., groups of two or more potentially immunogenic or immune response stimulating peptides which can be joined together in various arrangements (e.g., concatenated, overlapping).
  • the polytope (or nucleic acid encoding the polytope) can be administered in a standard immunization protocol, e.g., to animals, to test the effectiveness of the polytope in stimulating, enhancing and/or provoking an immune response.
  • polytopes can be joined together directly or via the use of flanking sequences to form polytopes, and the use of polytopes as vaccines is well known in the art (see, e.g., Thomson et al., Proc. Natl. Acad. Sci USA 92(13):5845-5849, 1995; Gilbert et al., Nature Biotechnol. 15(12):1280-1284, 1997; Thomson et al., J Immunol. 157(2):822-826, 1996; Tarn et al., J Exp. Med. 171(l):299-306, 1990).
  • Polytopes containing various numbers and combinations of epitopes can be prepared and tested for recognition by CTLs and for efficacy in increasing an immune response.
  • the peptides of the present invention can also be linked to other substances, so long as the resulting linked peptide retain the requisite CTL inducibility of the original peptide.
  • suitable substances include: for example, peptides, lipids, sugar and sugar chains, acetyl groups, natural and synthetic polymers, etc.
  • the peptides may contain modifications such as glycosylation, side chain oxidation, or phosphorylation, etc., provided that the modifications do not destroy the biological activity of the original peptide. These kinds of modifications may be performed to confer additional functions (e.g., targeting function, and delivery function) or to stabilize the polypeptide.
  • polypeptides For example, to increase the in vivo stability of a polypeptide, it is known in the art to introduce D-amino acids, amino acid mimetics or unnatural amino acids; this concept may also be adapted to the present polypeptides.
  • the stability of a polypeptide may be assayed in a number of ways. For instance, peptidases and various biological media, such as human plasma and serum, can be used to test stability (see, e.g., Verhoef et al., Eur J Drug Metab Pharmacokin 1986, 11: 291-302).
  • modified peptides that are substituted, deleted and/or added by one, two or several amino acid residues
  • those having same or higher activity as compared to original peptides can be screened for or selected.
  • the present invention also provides the method of screening for or selecting modified peptides having same or higher activity as compared to originals.
  • An illustrative method may include the steps of: a: substituting, deleting or adding at least one amino acid residue of a peptide of the present invention, b: determining the activity of the peptide produced in the step (a), and c: selecting the peptide having same or higher activity as compared to the original.
  • the activity to be assayed may include MHC binding activity, APC or CTL inducibility and cytotoxic activity.
  • the activity to be assayed is CTL inducibility and such activity can be assayed using the methods described in "EXAMPLES”
  • the peptides of the present invention may also be described as “TTK peptide(s)” or “TTK polypeptide(s)”.
  • TTK peptides may be prepared using well known techniques.
  • the peptides may be prepared synthetically, using recombinant DNA technology or chemical synthesis.
  • the peptides of the present invention may be synthesized individually or as longer polypeptides including two or more peptides.
  • the peptides may then be isolated, i.e., purified or isolated so as to be substantially free from other naturally occurring host cell proteins and fragments thereof, or any other chemical substances.
  • the peptides of the present invention may contain modifications, such as glycosylation, side chain oxidation, or phosphorylation, provided such modifications do not destroy the biological activity of the original peptide.
  • Other illustrative modifications include incorporation of D-amino acids or other amino acid mimetics that may be used, for example, to increase the serum half life of the peptides.
  • a peptide of the present invention may be obtained through chemical synthesis based on the selected amino acid sequence.
  • Examples of conventional peptide synthesis methods that may be adapted for the synthesis include: (i) Peptide Synthesis, Interscience, New York, 1966; (ii) The Proteins, Vol. 2, Academic Press, New York, 1976; (iii) Peptide Synthesis (in Japanese), Maruzen Co., 1975; (iv) Basics and Experiment of Peptide Synthesis (in Japanese), Maruzen Co., 1985; (v) Development of Pharmaceuticals (second volume) (in Japanese), Vol. 14 (peptide synthesis), Hirokawa, 1991; (vi) WO99/67288; and (vii) Barany G. & Merrifield R.B., Peptides Vol. 2, "Solid Phase Peptide Synthesis", Academic Press, New York, 1980, 100-118.
  • the present peptides may be obtained adapting any known genetic engineering method for producing peptides (e.g., Morrison J, J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (eds. Wu et al.) 1983, 101: 347-62).
  • a suitable vector harboring a polynucleotide encoding the objective peptide in an expressible form e.g., downstream of a regulatory sequence corresponding to a promoter sequence
  • the host cell is then cultured to produce the peptide of interest.
  • the peptide may also be produced in vitro adopting an in vitro translation system.
  • polynucleotides which encode any of the aforementioned peptides of the present invention. These include polynucleotides derived from the natural occurring TTK gene (GenBank Accession No. NM_003318 (for example, SEQ ID NO: 39)) as well as those having a conservatively modified nucleotide sequences thereof.
  • the phrase "conservatively modified nucleotide sequence” refers to sequences which encode identical or essentially identical amino acid sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein.
  • the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine.
  • the codon may be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a peptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid may be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a peptide is implicitly described in each disclosed sequence.
  • the polynucleotide of the present invention may be composed of DNA, RNA, or derivatives thereof.
  • a DNA molecule is composed of bases such as the naturally occurring bases A, T, C, and G, and T is replaced by U in an RNA.
  • bases such as the naturally occurring bases A, T, C, and G, and T is replaced by U in an RNA.
  • non-naturally occurring bases be included in polynucleotides, as well.
  • the polynucleotide of the present invention may encode multiple peptides that contain a peptide of the present invention and other epitope peptides with or without intervening amino acid sequences.
  • the intervening amino acid sequence may provide a cleavage site (e.g., enzyme recognition sequence) of the polynucleotide or the translated peptides.
  • the polynucleotide may include any additional sequences to the coding sequence encoding the peptide of the present invention.
  • the polynucleotide may be a recombinant polynucleotide that includes regulatory sequences required for the expression of the peptide or may be an expression vector (plasmid) with marker genes and such.
  • such recombinant polynucleotides may be prepared by the manipulation of polynucleotides through conventional recombinant techniques using, for example, polymerases and endonucleases.
  • a polynucleotide may be produced by insertion into an appropriate vector, which may be expressed when transfected into a competent cell.
  • a polynucleotide may be amplified using PCR techniques or expression in suitable hosts (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1989).
  • a polynucleotide may be synthesized using the solid phase techniques, as described in Beaucage SL & Iyer RP, Tetrahedron 1992, 48: 2223-311; Matthes et al., EMBO J 1984, 3: 801-5.
  • Exosomes The present invention further provides intracellular vesicles called exosomes, which present complexes formed between the peptides of the present invention and HLA antigens on their surface. Exosomes may be prepared, for example, using the methods detailed in Japanese Patent Application Kohyo Publications No. Hei 11-510507 and WO99/03499, and may be prepared using APCs obtained from patients who are subject to treatment and/or prevention. The exosomes of the present invention may be inoculated as vaccines, in a fashion similar to the peptides of the present invention.
  • HLA antigens included in the complexes must match that of the subject requiring treatment and/or prevention.
  • HLA-A2 particularly HLA-A*0201 and HLA-A*0206
  • A24 type or the A2 type that is highly expressed among the Japanese and Caucasian is favorable for obtaining effective results, and subtypes such as A*0201 and A*0206 also find use.
  • the type of HLA antigen of the patient requiring treatment is investigated in advance, which enables appropriate selection of peptides having high levels of binding affinity to the particular antigen, or having CTL inducibility by antigen presentation.
  • substitution, insertion, deletion, and/or addition of 1, 2, or several amino acids may be performed based on the amino acid sequence of the naturally occurring TTK partial peptide.
  • peptides having a amino acid sequence of SEQ ID NO: 3 may be preferably used as peptides to be presented by exosomes, and such peptides have high binding affinity with HLA-A2 such as HLA-A*0201.
  • the exosomes of the present invention present complexes formed between the peptide having an amino acid sequence of SEQ ID NO: 3 and HLA antigens on their surface.
  • the present invention also provides isolated antigen-presenting cells (APCs) that present complexes formed with HLA antigens and the peptides of the present invention on its surface.
  • the APCs may be derived from patients who are subject to treatment and/or prevention, and may be administered as vaccines by themselves or in combination with other drugs including the peptides of the present invention, exosomes, or CTLs.
  • the APCs are not limited to a particular kind of cells and include dendritic cells (DCs), Langerhans cells, macrophages, B cells, and activated T cells, which are known to present proteinaceous antigens on their cell surface so as to be recognized by lymphocytes. Since DC is a representative APC having the strongest CTL inducing activity among APCs, DCs find use as the APCs of the present invention.
  • the APCs of the present invention may be obtained by inducing DCs from peripheral blood monocytes and then contacting (stimulating) them with the peptides of the present invention in vitro, ex vivo or in vivo.
  • the peptides of the present invention are administered to the subjects, APCs that present the peptides of the present invention are induced in the body of the subject. Therefore, the APCs of the present invention may be obtained by collecting the APCs from the subject after administering the peptides of the present invention to the subject.
  • the APCs of the present invention may be obtained by contacting APCs collected from a subject with the peptide of the present invention.
  • the APCs of the present invention may be administered to a subject for inducing immune response against cancer in the subject by themselves or in combination with other drugs including the peptides, exosomes or CTLs of the present invention.
  • the ex vivo administration may include steps of: a: collecting APCs from a first subject, b: contacting with the APCs of step a, with the peptide, and c: administering the APCs of step b to a second subject.
  • the first subject and the second subject may be the same individual, or may be different individuals.
  • use of the peptides of the present invention for manufacturing a pharmaceutical composition inducing antigen-presenting cells is provided.
  • the present invention provides a method or process for manufacturing a pharmaceutical composition inducing antigen-presenting cells.
  • the present invention also provides the peptides of the present invention for inducing antigen-presenting cells.
  • the APCs obtained by step b may be a vaccine for treating and/or preventing cancer, including, but are not limited to, lung cancer, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophageal cancer, gastric cancer, diffused type gastric cancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma, SCLC, soft tissue tumor and testicular tumor.
  • cancer including, but are not limited to, lung cancer, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophageal cancer, gastric cancer, diffused type gastric cancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma, SCLC, soft tissue tumor and testicular tumor.
  • the APCs have a high level of CTL inducibility.
  • high level of CTL inducibility the high level is relative to the level of that by APC contacting with no peptide or peptides which may not induce the CTL.
  • Such APCs having a high level of CTL inducibility may be prepared by a method that includes the step of transferring a polynucleotide encoding the peptide of the present invention to APCs in vitro as well as the method mentioned above.
  • the introduced genes may be in the form of DNAs or RNAs.
  • Examples of methods for introduction include, without particular limitations, various methods conventionally performed in this field, such as lipofection, electroporation, or calcium phosphate method may be used. More specifically, it may be performed as described in Cancer Res 1996, 56: 5672-7; J Immunol 1998, 161: 5607-13; J Exp Med 1996, 184: 465-72; Published Japanese Translation of International Publication No. 2000-509281.
  • the gene undergoes transcription, translation, and such in the cell, and then the obtained protein is processed by MHC Class I or Class II, and proceeds through a presentation pathway to present partial peptides.
  • the APCs of the present invention may be those that present complexes formed between HLA antigens and the peptides having an amino acid sequence of SEQ ID NO: 3 on its surface. More preferably, APCs carry HLA-A2 antigen such as HLA-A*0201, and present complexes formed with such HLA-A2 antigens and the peptides of the present invention (e.g., peptides having an amino acid sequence of SEQ ID NO: 3) on its surface.
  • HLA-A2 antigen such as HLA-A*0201
  • CTLs Cytotoxic T lymphocytes
  • a CTL induced against any one of the peptides of the present invention strengthens the immune response targeting cancer cells in vivo and thus may be used as vaccines, in a fashion similar to the peptides per se.
  • the present invention provides isolated CTLs that are specifically induced or activated by any one of the present peptides.
  • Such CTLs may be obtained by (1) administering the peptide(s) of the present invention to a subject or (2) contacting (stimulating) subject-derived APCs, and CD8 positive cells, or peripheral blood mononuclear leukocytes in vitro with the peptide(s) of the present invention or (3) contacting CD8 positive cells or peripheral blood mononuclear leukocytes in vitro with the APCs or exosomes presenting a complex of an HLA antigen and the peptide on its surface or (4) introducing a gene that includes a polynucleotide encoding a T cell receptor (TCR) subunit capable of binding to the peptide of the present invention.
  • TCR T cell receptor
  • the CTLs of the present invention may be derived from patients who are subject to treatment and/or prevention, and may be administered by themselves or in combination with other drugs including the peptides of the present invention or exosomes for the purpose of regulating effects.
  • the obtained CTLs act specifically against target cells presenting the peptides of the present invention, for example, the same peptides used for induction.
  • the target cells may be cells that endogenously express TTK, such as cancer cells, or cells that are transfected with the TTK gene; and cells that present a peptide of the present invention on the cell surface due to stimulation by the peptide may also serve as targets of activated CTL attack.
  • T cell receptor The present invention also provides a composition including nucleic acids encoding polypeptides that are capable of forming a subunit of a T cell receptor (TCR), and methods of using the same.
  • the TCR subunits of the present invention have the ability to form TCRs that confer specificity to T cells against tumor cells presenting TTK.
  • the nucleic acids encoding alpha- and beta- chains that constitute the TCR subunits of the CTL induced with one or more peptides of the present invention may be identified (WO2007/032255 and Morgan et al., J Immunol, 171, 3288 (2003)).
  • PCR methods are preferred to analyze the nucleotide sequences encoding TCR subunits.
  • the PCR primers for the analysis can be, for example, 5'-R primers (5'-gtctaccaggcattcgcttcat-3') as 5' side primers (SEQ ID NO: 41) and 3-TRa-C primers (5'-tcagctggaccacagccgcagcgt-3') specific to TCR alpha chain C region (SEQ ID NO: 42), 3-TRb-C1 primers (5'-tcagaaatcctttctctttgac-3') specific to TCR beta chain C1 region (SEQ ID NO: 43) or 3-TRbeta-C2 primers (5'- ctagcctctggaatcctttctcttt-3') specific to TCR beta chain C2 region (SEQ ID NO: 44) as 3' side primers, but not limited thereto.
  • the nucleic acids encoding the TCR subunits may be incorporated into suitable vectors, e.g., retroviral vectors. These vectors are well known in the art.
  • the nucleic acids or the vectors including them usefully may be transferred into a T cell, for example, a T cell from a patient.
  • the present invention provides an off-the-shelf composition allowing rapid modification of a patient's own T cells (or those of another mammal) to rapidly and easily produce modified T cells having excellent cancer cell killing properties.
  • the specific TCR is a receptor capable of specifically recognizing a complex of a peptide of the present invention and HLA molecule, giving a T cell specific activity against the target cell when the TCR is presented on the surface of the T cell.
  • a specific recognition of the above complex may be confirmed by any known methods, preferred examples of which include HLA multimer staining analysis using HLA molecules and peptides of the present invention, and ELISPOT assay. By performing the ELISPOT assay, it can be confirmed whether a T cell transduced with the nucleic acid encoding the TCR subunits recognizes a cell expressing HLA molecule and TTK, and the signal is transmitted intracellularly.
  • the TCR subunits introduced into a T cell can give a T cell cytotoxic activity by known methods in the art.
  • Preferred methods include, for example, chromium release assay using HLA-A2 positive and TTK over-expressing cells.
  • the present invention provides CTLs which are prepared by transduction with the nucleic acids encoding the TCR subunits that bind to a complex formed between the peptide of the present invention and HLA-A2 molecule such as HLA-A*0201.
  • the transduced CTLs are capable of homing to cancer cells in vivo, and may be expanded by well known culturing methods in vitro (e.g., Kawakami et al., J Immunol., 142, 3452-3461 (1989)).
  • the CTLs of the present invention may be used to form an immunogenic composition useful in treating and/or preventing cancer in a patient in need of therapy or protection (WO2006/031221).
  • TTK expression is specifically elevated in cancers, examples of which include, but are not limited to, lung cancer, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophageal cancer, gastric cancer, diffused type gastric cancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma, SCLC, soft tissue tumor and testicular tumor as compared with normal tissue, the peptides of the present invention or polynucleotides encoding such peptides may be used for the treatment and/or prophylaxis of cancer, and/or for preventing the postoperative recurrence thereof.
  • the present invention provides a pharmaceutical agent or composition for treating and/or preventing the postoperative recurrence thereof, such agent or composition including as an active ingredient one or more of the peptides, or polynucleotides of the present invention.
  • the present peptides may be expressed on the surface of any of the foregoing exosomes or cells, such as APCs for the use as pharmaceutical agents or compositions.
  • the aforementioned CTLs which target any one of the peptides of the present invention may also be used as the active ingredient of the present pharmaceutical agents or compositions.
  • the pharmaceutical agents and compositions of the present invention can also find use as a vaccine.
  • the phrase "vaccine” refers to a substance that has the function to induce anti-tumor immunity upon inoculation into animals.
  • the pharmaceutical agents or compositions of the present invention can be used to treat and/or prevent cancers, and/or prevention of postoperative recurrence thereof in subjects or patients including human and any other mammal including, but not limited to, mouse, rat, guinea-pig, rabbit, cat, dog, sheep, goat, pig, cattle, horse, monkey, baboon, and chimpanzee, particularly a commercially important animal or a domesticated animal.
  • the present invention also provides the use of an active ingredient selected from among: (a) a peptide of the present invention; (b) a nucleic acid encoding such a peptide as disclosed herein in an expressible form; (c) an APC or an exosome presenting a peptide of the present invention on its surface; and (d) a cytotoxic T cell of the present invention in manufacturing a pharmaceutical agent or composition for treating or preventing cancer or tumor.
  • an active ingredient selected from among: (a) a peptide of the present invention; (b) a nucleic acid encoding such a peptide as disclosed herein in an expressible form; (c) an APC or an exosome presenting a peptide of the present invention on its surface; and (d) a cytotoxic T cell of the present invention in manufacturing a pharmaceutical agent or composition for treating or preventing cancer or tumor.
  • the present invention further provides an active ingredient selected from among: (a) a peptide of the present invention; (b) a nucleic acid encoding such a peptide as disclosed herein in an expressible form; (c) an APC or an exosome presenting a peptide of the present invention on its surface; and (d) a cytotoxic T cell of the present invention for use in the treatment or prevention of cancer or tumor.
  • an active ingredient selected from among: (a) a peptide of the present invention; (b) a nucleic acid encoding such a peptide as disclosed herein in an expressible form; (c) an APC or an exosome presenting a peptide of the present invention on its surface; and (d) a cytotoxic T cell of the present invention for use in the treatment or prevention of cancer or tumor.
  • the present invention further provides a method or process for manufacturing a pharmaceutical agent or composition for treating or preventing cancer or tumor, wherein the method or process includes the step of formulating a pharmaceutically or physiologically acceptable carrier with an active ingredient selected from among: (a) a peptide of the present invention; (b) a nucleic acid encoding such a peptide as disclosed herein in an expressible form; (c) an APC or an exosome presenting a peptide of the present invention on its surface; and (d) a cytotoxic T cell of the present invention as active ingredients.
  • a pharmaceutically or physiologically acceptable carrier with an active ingredient selected from among: (a) a peptide of the present invention; (b) a nucleic acid encoding such a peptide as disclosed herein in an expressible form; (c) an APC or an exosome presenting a peptide of the present invention on its surface; and (d) a cytotoxic T cell of the present invention as active ingredients.
  • the present invention also provides a method or process for manufacturing a pharmaceutical agent or composition for treating or preventing cancer or tumor, wherein the method or process includes the steps of admixing an active ingredient with a pharmaceutically or physiologically acceptable carrier, wherein the active ingredient is selected from among: (a) a peptide of the present invention; (b) a nucleic acid encoding such a peptide as disclosed herein in an expressible form; (c) an APC or an exosome presenting a peptide of the present invention on its surface; and (d) a cytotoxic T cell of the present invention.
  • peptides having an amino acid sequence of SEQ ID NOs: 3 have been found to be HLA-A2 restricted epitope peptides or the candidates that may induce potent and specific immune response. Therefore, the present pharmaceutical agnets or compositions which include at least one peptide with the amino acid sequences of SEQ ID NOs: 3 are particularly suited for the administration to subjects whose HLA-A antigen is HLA-A2. The same applies to pharmaceutical agents or compositions that include polynucleotides encoding any of these peptides (i.e., the polynucleotides of the present invention).
  • Cancers to be treated by the pharmaceutical agents or compositions of the present invention are not limited and include any cancer in which TTK is involved (e.g., is overexpressed), including, for example, lung cancer, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophageal cancer, gastric cancer, diffused type gastric cancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma, SCLC, soft tissue tumor and testicular tumor.
  • any cancer in which TTK is involved e.g., is overexpressed
  • lung cancer bladder cancer
  • breast cancer cervical cancer
  • CML colorectal cancer
  • endometriosis esophageal cancer
  • gastric cancer diffused type gastric cancer
  • liver cancer NSCLC
  • lymphoma lymphoma
  • osteosarcoma ovarian cancer
  • pancreatic cancer prostate cancer
  • SCLC soft tissue
  • the present pharmaceutical agents or compositions may contain in addition to the aforementioned active ingredients, other peptides which have the ability to induce CTLs against cancerous cells, other polynucleotides encoding the other peptides, other cells that present the other peptides, or such.
  • the other peptides that have the ability to induce CTLs against cancerous cells are exemplified by cancer specific antigens (e.g., identified TAAs), but are not limited thereto.
  • the pharmaceutical agents or compositions of the present invention may optionally include other therapeutic substances as an active ingredient, so long as the substance does not inhibit the antitumoral effect of the active ingredient, e.g., any of the present peptides.
  • formulations may include anti-inflammatory agents, pain killers, chemotherapeutics, and the like.
  • the medicaments of the present invention may also be administered sequentially or concurrently with the one or more other pharmacologic agents.
  • the amounts of medicament and pharmacologic agent depend, for example, on what type of pharmacologic agent(s) is/are used, the disease being treated, and the scheduling and routes of administration. It should be understood that in addition to the ingredients particularly mentioned herein, the pharmaceutical agents or compositions of the present invention may include other agents conventional in the art having regard to the type of formulation in question.
  • the present pharmaceutical agents or compositions may be included in articles of manufacture and kits containing materials useful for treating the pathological conditions of the disease to be treated, e.g., cancer.
  • the article of manufacture may include a container of any of the present pharmaceutical substances or compositions with a label. Suitable containers include bottles, vials, and test tubes. The containers may be formed from a variety of materials, such as glass or plastic.
  • the label on the container should indicate the substance or composition is used for treating or prevention of one or more conditions of the disease. The label may also indicate directions for administration and so on.
  • a kit including a pharmaceutical agent or composition of the present invention may optionally further include a second container housing a pharmaceutically-acceptable diluent.
  • compositions can, if desired, be presented in a pack or dispenser device which can contain one or more unit dosage forms containing the active ingredient.
  • the pack can, for example, include metal or plastic foil, such as a blister pack.
  • the pack or dispenser device can be accompanied by instructions for administration.
  • compositions containing the peptides as the active ingredient can be administered directly as a pharmaceutical agent or composition, or if necessary, may be formulated by conventional formulation methods.
  • carriers, excipients, and such that are ordinarily used for drugs can be included as appropriate without particular limitations. Examples of such carriers are sterilized water, physiological saline, phosphate buffer, culture fluid and such.
  • the pharmaceutical agents or compositions can contain as necessary, stabilizers, suspensions, preservatives, surfactants and such.
  • the pharmaceutical agents or compositions of the present invention can be used for anticancer purposes.
  • the peptides of the present invention can be prepared as a combination composed of two or more of peptides of the present invention and other epitope peptides (e.g., peptides derived from other TAAs), to induce CTL in vivo.
  • the peptide combination can take the form of a cocktail or can be conjugated to each other using standard techniques.
  • the peptides can be chemically linked or expressed as a single fusion polypeptide sequence that may have one or several amino acid(s) as a linker (e.g., Lysine linker: K. S. Kawamura et al. J. Immunol. 2002, 168: 5709-5715).
  • the peptides in the combination can be the same or different.
  • the peptides are presented in high density by the HLA antigens on APCs, then CTLs that specifically react toward the complex formed between the displayed peptide and the HLA antigen are induced.
  • APCs e.g., DCs
  • APCs are removed from subjects and then stimulated by the peptides of the present invention to obtain APCs that present any of the peptides of the present invention on their cell surface.
  • the pharmaceutical agents or compositions for the treatment and/or prevention of cancer can also include an adjuvant known to effectively establish cellular immunity.
  • the pharmaceutical compositions can be administered with other active ingredients or administered by formulation into granules.
  • An adjuvant refers to any compound, substance or composition that enhances the immune response against the protein when administered together (or successively) with the protein having immunological activity. Adjuvants contemplated herein include those described in the literature (Clin Microbiol Rev 1994, 7: 277-89).
  • Suitable adjuvants include, but are not limited to, aluminum phosphate, aluminum hydroxide, alum, cholera toxin, salmonella toxin, Incomplete Freund's adjuvant (IFA), Complete Freund's adjuvant (CFA), ISCOMatrix, GM-CSF, CpG, O/W emulsion, and the like.
  • IFA Incomplete Freund's adjuvant
  • CFA Complete Freund's adjuvant
  • ISCOMatrix GM-CSF
  • CpG CpG
  • O/W emulsion examples of suitable adjuvants
  • liposome formulations, granular formulations in which the peptide is bound to few-micrometers diameter beads, and formulations in which a lipid is bound to the peptide may be conveniently used.
  • the pharmaceutical agents or compositions of the present invention may further include a component that primes CTL.
  • Lipids have been identified as agents capable of priming CTL in vivo against viral antigens.
  • palmitic acid residues can be attached to the epsilon- and alpha-amino groups of a lysine residue and then linked to a peptide of the present invention.
  • the lipidated peptide can then be administered either directly in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant.
  • lipid priming of CTL responses E.
  • compositions containing polynucleotides as active ingredient can also include nucleic acids encoding the peptide(s) disclosed herein in an expressible form.
  • the phrase "in an expressible form” means that the polynucleotide, when introduced into a cell, will be expressed in vivo as a polypeptide that induces anti-tumor immunity.
  • the nucleic acid sequence of the polynucleotide of interest includes regulatory elements necessary for expression of the polynucleotide.
  • the polynucleotide(s) can be equipped so to achieve stable insertion into the genome of the target cell (see, e.g., Thomas KR & Capecchi MR, Cell 1987, 51: 503-12 for a description of homologous recombination cassette vectors. See also, e.g., Wolff et al., Science 1990, 247: 1465-8; U.S. Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720).
  • DNA-based delivery technologies include "naked DNA”, facilitated (bupivacaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., U.S. Patent No. 5,922,687).
  • the peptides of the present invention can also be expressed by viral or bacterial vectors.
  • expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus, e.g., as a vector to express nucleotide sequences that encode the peptide. Upon introduction into a host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits an immune response.
  • Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin).
  • any of the aforementioned pharmaceutical agents or compositions of the present invention can be used for inducing CTLs, and in addition thereto, those including the peptides and polynucleotides can be also be used for inducing APCs as discussed below.
  • the present invention provides methods of inducing APCs with high CTL inducibility using the peptides or polynucleotides of the present invention.
  • the methods of the present invention include the step of contacting APCs with the peptides of the present invention in vitro, ex vivo or in vivo.
  • the method contacting APCs with the peptides ex vivo or in vitro can include steps of: a: collecting APCs from a subject:, and b: contacting the APCs of step a with the peptide.
  • the APCs are not limited to a particular kind of cells and include DCs, Langerhans cells, macrophages, B cells, and activated T cells, which are known to present proteinaceous antigens on their cell surface so as to be recognized by lymphocytes.
  • DCs can be used since they have the strongest CTL inducibility among APCs.
  • Any peptides of the present invention can be used by themselves or with other peptides.
  • the present invention may also include introducing the polynucleotide of the present invention into an APCs to induce APCs with CTL inducibility.
  • the method can include steps of: a: collecting APCs from a subject:, and b: introducing a polynucleotide encoding the peptide of the present invention. Step b can be performed as described above in section "VI. Antigen-presenting cells”.
  • the present invention provides a method for preparing an antigen-presenting cell (APC) which specifically induces CTL activity against TTK, wherein the method can include one of the following steps: (a) contacting an APC with a peptide of the present invention in vitro, ex vivo or in vivo; and (b) introducing a polynucleotide encoding a peptide of the present invention into an APC.
  • APC antigen-presenting cell
  • the methods for inducing CTLs may include at least one step selected from the group consisting of: a) contacting a CD8 positive T cell with an antigen-presenting cell and/or an exosome that presents on its surface a complex of an HLA antigen and a peptide of the preset invention; and b) introducing a polynucleotide encoding a polypeptide that is capable of forming a TCR subunit recognizing a complex of a peptide of the present invention and an HLA antigen into a CD8 positive cell.
  • the methods of the present invention includes the step of administering the peptides, the polynucleotides, the APCs or exosomes of the present invention to a subject.
  • CTLs can be also induced by using them ex vivo or in vitro, and after inducing CTL, the activated CTLs can be returned to the subject.
  • the APCs to be co-cultured with the CD8 positive cells in above step c can also be prepared by transferring a gene that includes a polynucleotide of the present invention into APCs as described above in section "VI. Antigen-presenting cells"; though the present invention is not limited thereto, and encompasses any APC that effectively presents on its surface a complex of an HLA antigen and a peptide of the present invention.
  • the exosomes that presents on its surface a complex of an HLA antigen and the peptide of the present invention can be also used.
  • the present invention can include the step of co-culturing exosomes presenting on its surface a complex of an HLA antigen and the peptide of the present invention.
  • exosomes can be prepared by the methods described above in section "V. Exosomes”.
  • the methods of the present invention may include the step of administering agent(s) or composition(s) containing any of the peptides of the present invention or polynucleotides encoding them.
  • inventive methods also contemplate the administration of exosomes or APCs presenting any of the peptides of the present invention.
  • IX Pharmaceutical agents or compositions
  • pharmaceutical agents or compositions particularly the part describing the use of the pharmaceutical agents or compositions of the present invention as vaccines.
  • the exosomes and APCs that can be employed for the present methods for inducing immune response are described in detail under the items of "V. Exosomes", “VI. Antigen-presenting cells (APCs)", and (1) and (2) of "X.
  • the present invention also provides a method or process for manufacturing a pharmaceutical agent or composition inducing immune response, wherein the method may include the step of admixing or formulating the peptide of the present invention with a pharmaceutically acceptable carrier.
  • the method of the present invention may include the step of administrating a vaccine or a pharmaceutical agent or composition that contains: (a) a peptide of the present invention; (b) a nucleic acid encoding such a peptide as disclosed herein in an expressible form; (c) an APC or an exosome presenting a peptide of the present invention on its surface; or (d) a cytotoxic T cell of the present invention.
  • the present invention provides a method for treating cancer (over)expressing TTK in a patient in need thereof, such method may include the steps of: i) determining the expression level of TTK in cells or tissue(s) obtained from a subject with the cancer to be treated; ii) comparing the expression level of TTK with normal control level; and iii) administrating at least one component selected from the group consisting of (a) to (d) described above to a subject with cancer overexpressing TTK compared with normal control.
  • any subject-derived cell or tissue can be used for the determination of TTK expression so long as it includes the objective transcription or translation product of TTK.
  • suitable samples include, but are not limited to, bodily tissues and fluids, such as blood, sputum and urine.
  • the subject-derived cell or tissue sample contains a cell population including an epithelial cell, more preferably a cancerous epithelial cell or an epithelial cell derived from tissue suspected to be cancerous. Further, if necessary, the cell may be purified from the obtained bodily tissues and fluids, and then used as the subjected-derived sample.
  • a control level determined from a biological sample that is known to be non-cancerous is referred to as a "normal control level”.
  • the control level is determined from a cancerous biological sample, it is referred to as a "cancerous control level”.
  • Difference between a sample expression level and a control level can be normalized to the expression level of control nucleic acids, e.g., housekeeping genes, whose expression levels are known not to differ depending on the cancerous or non-cancerous state of the cell.
  • Exemplary control genes include, but are not limited to, beta-actin, glyceraldehyde 3 phosphate dehydrogenase, and ribosomal protein P1.
  • a subject to be treated by the present method is preferably a mammal.
  • Exemplary mammals include, but are not limited to, e.g., human, non-human primate, mouse, rat, dog, cat, horse, and cow.
  • the Tm is the temperature (under a defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to their target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 degrees C for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60 degrees C for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing substances, such as formamide.
  • the subject may be diagnosed with cancer to be treated.
  • the present invention also provides a method of (i) diagnosing whether a subject suspected to have cancer to be treated, and/or (ii) selecting a subject for cancer treatment, which method may include the steps of: a) determining the expression level of TTK in cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated; b) comparing the expression level of TTK with a normal control level; c) diagnosing the subject as having the cancer to be treated, if the expression level of TTK is increased as compared to the normal control level; and d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).
  • such a method may include the steps of: a) determining the expression level of TTK in cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated; b) comparing the expression level of TTK with a cancerous control level; c) diagnosing the subject as having the cancer to be treated, if the expression level of TTK is similar or equivalent to the cancerous control level; and d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).
  • the reagent when the reagent is a probe against the TTK mRNA, the reagent may be immobilized on a solid matrix, such as a porous strip, to form at least one detection site.
  • the measurement or detection region of the porous strip may include a plurality of sites, each containing a nucleic acid (probe).
  • a test strip may also contain sites for negative and/or positive controls. Alternatively, control sites may be located on a strip separated from the test strip.
  • the different detection sites may contain different amounts of immobilized nucleic acids, i.e., a higher amount in the first detection site and lesser amounts in subsequent sites.
  • the number of sites displaying a detectable signal provides a quantitative indication of the amount of TTK mRNA present in the sample.
  • the detection sites may be configured in any suitably detectable shape and are typically in the shape of a bar or dot spanning the width of a test strip.
  • the kit of the present invention may further include a positive control sample or TTK standard sample.
  • the positive control sample of the present invention may be prepared by collecting TTK positive samples and then assaying their TTK levels.
  • a purified TTK protein or polynucleotide may be added to cells that do not express TTK to form the positive sample or the TTK standard sample.
  • purified TTK may be a recombinant protein.
  • the TTK level of the positive control sample is, for example, more than the cut off value.
  • the present invention further provides a diagnostic kit including, a protein or a partial protein thereof capable of specifically recognizing the antibody of the present invention or an immunogenic fragment thereof.
  • partial peptides and immunogenic fragment of the proteins of the present invention contemplated herein include polypeptides composed of at least 8, preferably 15, and more preferably 20 contiguous amino acids in the amino acid sequence of the protein of the present invention.
  • Cancer can be diagnosed by detecting an antibody in a sample (e.g., blood, tissue) using a protein or a peptide (polypeptide) of the present invention. Methods for preparing a peptide or protein of the present invention are as described above.
  • the methods for diagnosing cancer of the present invention can be performed by determining the difference between the amount of anti-TTK antibody and that in the corresponding control sample as describe above.
  • the subject is suspected to be suffering from cancer, if cells or tissues of the subject contain antibodies against the expression products (TTK) of the gene and the quantity of the anti-TTK antibody is determined to be more than the cut off value in level compared to that in normal control.
  • TTK expression products
  • the present invention further provides methods or diagnostic reagents for evaluating the immunological response of subject using peptide epitopes as described herein.
  • HLA restricted peptides as described herein may be used as reagents for evaluating or predicting an immune response of a subject.
  • the immune response to be evaluated may be induced by contacting an immunogen with immunocompetent cells in vitro or in vivo.
  • the substances or compositions employed as the reagent may be composition that may result in the production of antigen specific CTLs that recognize and bind to the peptide epitope(s).
  • the peptide reagents need not be used as the immunogen.
  • a peptide that binds to an HLA molecule is refolded in the presence of the corresponding HLA heavy chain and beta 2-microglobulin to generate a trimolecular complex.
  • carboxyl terminal of the heavy chain is biotinylated at a site that was previously engineered into the protein.
  • streptavidin is added to the complex to form tetramer consisting of the trimolecular complex and streptavidin.
  • the tetramer can be used to stain antigen specific cells.
  • the cells can then be identified, for example, by flow cytometry. Such an analysis may be used for diagnostic or prognostic purposes. Cells identified by the procedure can also be used for therapeutic purposes.
  • the present invention also provides reagents to evaluate immune recall responses (see, e.g., Bertoni et al, J. Clin. Invest. 100: 503-513, 1997 and Penna et al., J Exp. Med. 174: 1565-1570, 1991) including peptides of the present invention.
  • patient PBMC samples from individuals with cancer to be treated can be analyzed for the presence of antigen-specific CTLs using specific peptides.
  • a blood sample containing mononuclear cells can be evaluated by cultivating the PBMCs and stimulating the cells with a peptide of the present invention. After an appropriate cultivation period, the expanded cell population can be analyzed, for example, for CTL activity.
  • the peptides may also be used as reagents to evaluate the efficacy of a vaccine.
  • PBMCs obtained from a patient vaccinated with an immunogen may be analyzed using, for example, either of the methods described above.
  • the patient is HLA typed, and peptide epitope reagents that recognize the allele specific molecules present in the patient are selected for the analysis.
  • the immunogenicity of the vaccine may be indicated by the presence of epitope-specific CTLs in the PBMC sample.
  • the peptides of the present invention may also be used to make antibodies, using techniques well known in the art (see, e.g., CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY; and Antibodies A Laboratory Manual, Harlow and Lane, Cold Spring Harbor Laboratory Press, 1989), which may find use as reagents to diagnose, detect or monitor cancer.
  • Such antibodies may include those that recognize a peptide in the context of an HLA molecule, i.e., antibodies that bind to a peptide-MHC complex.
  • the present invention provides a method for diagnosing or detecting a disorder characterized by expression of a TTK immunogenic polypeptide.
  • Such methods involve determining expression of a TTK HLA binding peptide, or a complex of a TTK HLA binding peptide and an HLA class I molecule in a biological sample.
  • the expression of a peptide or complex of peptide and HLA class I molecule can be determined or detected by assaying with a binding partner for the peptide or complex.
  • a binding partner for the peptide or complex may be an antibody recognizes and specifically bind to the peptide.
  • TTK tumor growth factor
  • a biological sample such as a tumor biopsy
  • TTK primers An example of tumor expression is presented herein and further disclosure of exemplary conditions and primers for TTK amplification can be found in WO2003/27322.
  • Multimer staining, intracellular lymphokine staining and ELISPOT assays all appear to be at least 10-fold more sensitive than more conventional assays (Murali-Krishna, K. et al., 1998, Immunity 8: 177; Lalvani, A. et al., 1997, J. Exp. Med. 186: 859; Dunbar, P. R. et al., 1998, Curr. Biol. 8: 413).
  • Pentamers e.g., US 2004-209295A
  • dextramers e.g., WO 02/072631
  • streptamers e.g., Nature medicine 6. 631-637 (2002)
  • intracellularly expressed antibodies may find therapeutical use in treating cancers in which the expression of TTK is involved, examples of which include, but are not limited to, lung cancer, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophageal cancer, gastric cancer, diffused type gastric cancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma, SCLC, soft tissue tumor and testicular tumor.
  • lung cancer e.g., bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophageal cancer, gastric cancer, diffused type gastric cancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma, SCLC, soft tissue tumor and testicular tumor.
  • the present invention also provides various immunological assay for the detection and/or quantification of the TTK protein (SEQ ID NO: 40) or fragments thereof including polypeptides having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 6, 15 and 22.
  • Such assays may include one or more anti-TTK antibodies capable of recognizing and binding a TTK protein or fragments thereof, as appropriate.
  • anti-TTK antibodies binding to TTK polypeptide preferably recognize polypeptide consisting of amino acid sequences selected from the group consisting of SEQ ID NOs: 1, 2, 3, 6, 15 and 22. A binding specificity of antibody can be confirmed with inhibition test.
  • immunological assays are performed within various immunological assay formats well known in the art, including but not limited to, various types of radioimmunoassays, immuno-chromatograph technique, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.
  • immunological but non-antibody assays of the present invention may also include T cell immunogenicity assays (inhibitory or stimulatory) as well as MHC binding assays.
  • the present invention contemplates immunological imaging methods capable of detecting cancers expressing TTK, examples of which include, but are not limited to, radioscintigraphic imaging methods using labeled antibodies of the present invention.
  • Such assays can find clinical use in the detection, monitoring, and prognosis of TTK expressing cancers, examples of which include, but are not limited to, lung cancer, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophageal cancer, gastric cancer, diffused type gastric cancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma, SCLC, soft tissue tumor and testicular tumor.
  • the peptide to be used as an immunization antigen may be a complete protein or a partial peptide of the protein.
  • a partial peptide may include, for example, the amino (N)-terminal or carboxy (C)-terminal fragment of a peptide of the present invention.
  • an antibody is defined as a protein that reacts with either the full length or a fragment of a TTK peptide.
  • an antibody of the present invention can recognize fragment peptides of TTK consisting of amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 6, 15 and 22.
  • Methods for synthesizing oligopeptide are well known in the arts. After the synthesis, peptides may be optionally purified prior to use as immunogen.
  • the oligopeptide e.g., 9- or 10mer
  • the oligopeptide e.g., 9- or 10mer
  • the oligopeptide may be conjugated or linked with carriers to enhance the immunogenicity.
  • Keyhole-limpet hemocyanin (KLH) is well known as the carrier. Method for conjugating KLH and peptide are also well known in the arts.
  • a gene encoding a peptide of the present invention or fragment thereof may be inserted into a known expression vector, which is then used to transform a host cell as described herein.
  • the desired peptide or fragment thereof may be recovered from the outside or inside of host cells by any standard method, and may subsequently be used as an antigen.
  • whole cells expressing the peptide or their lysates or a chemically synthesized peptide may be used as the antigen.
  • Any mammalian animal may be immunized with the antigen, but preferably the compatibility with parental cells used for cell fusion is taken into account. In general, animals of Rodentia, Lagomorpha or Primates may be used.
  • Animals of the family Rodentia include, for example, mouse, rat and hamster.
  • Animals of the family Lagomorpha include, for example, rabbit.
  • Animals of the Primate family include, for example, a monkey of Catarrhini (old world monkey) such as Macaca fascicularis, rhesus monkey, sacred baboon and chimpanzees.
  • antigens may be diluted and suspended in an appropriate amount of phosphate buffered saline (PBS), physiological saline, etc.
  • PBS phosphate buffered saline
  • the antigen suspension may be mixed with an appropriate amount of a standard adjuvant, such as Freund's complete adjuvant, made into emulsion and then administered to mammalian animals.
  • a standard adjuvant such as Freund's complete adjuvant
  • an appropriately amount of Freund's incomplete adjuvant every 4 to 21 days.
  • An appropriate carrier may also be used for immunization.
  • serum may be examined by a standard method for an increase in the amount of desired antibodies.
  • Polyclonal antibodies against the peptides of the present invention may be prepared by collecting blood from the immunized mammal examined for the increase of desired antibodies in the serum, and by separating serum from the blood by any conventional method.
  • Polyclonal antibodies may include serum containing the polyclonal antibodies, as well as the fraction containing the polyclonal antibodies may be isolated from the serum.
  • Immunoglobulin G or M can be prepared from a fraction which recognizes only the peptide of the present invention using, for example, an affinity column coupled with the peptide of the present invention, and further purifying this fraction using protein A or protein G column.
  • immune cells are collected from the mammal immunized with the antigen and checked for the increased level of desired antibodies in the serum as described above, and are subjected to cell fusion.
  • the immune cells used for cell fusion may preferably be obtained from spleen.
  • Other preferred parental cells to be fused with the above immunocyte include, for example, myeloma cells of mammalians, and more preferably myeloma cells having an acquired property for the selection of fused cells by drugs.
  • the above immunocyte and myeloma cells can be fused according to known methods, for example, the method of Milstein et al. (Galfre and Milstein, Methods Enzymol 73: 3-46 (1981)).
  • human lymphocytes such as those infected by EB virus may be immunized with a peptide, peptide expressing cells or their lysates in vitro. Then, the immunized lymphocytes are fused with human-derived myeloma cells that are capable of indefinitely dividing, such as U266, to yield a hybridoma producing a desired human antibody that is able to bind to the peptide can be obtained (Unexamined Published Japanese Patent Application No. Sho 63-17688).
  • the obtained hybridomas are subsequently transplanted into the abdominal cavity of a mouse and the ascites are extracted.
  • the obtained monoclonal antibodies can be purified by, for example, ammonium sulfate precipitation, a protein A or protein G column, DEAE ion exchange chromatography or an affinity column to which the peptide of the present invention is coupled.
  • the antibody of the present invention can be used not only for purification and detection of the peptide of the present invention, but also as a candidate for agonists and antagonists of the peptide of the present invention.
  • an immune cell such as an immunized lymphocyte, producing antibodies may be immortalized by an oncogene and used for preparing monoclonal antibodies.
  • an antibody of the present invention may be a fragment of an antibody or modified antibody, so long as it binds to one or more of the peptides of the present invention.
  • the antibody fragment may be Fab, F(ab') 2 , Fv or single chain Fv (scFv), in which Fv fragments from H and L chains are ligated by an appropriate linker (Huston et al., Proc Natl Acad Sci USA 85: 5879-83 (1988)). More specifically, an antibody fragment may be generated by treating an antibody with an enzyme, such as papain or pepsin.
  • a gene encoding the antibody fragment may be constructed, inserted into an expression vector and expressed in an appropriate host cell (see, for example, Co et al., J Immunol 152: 2968-76 (1994); Better and Horwitz, Methods Enzymol 178: 476-96 (1989); Pluckthun and Skerra, Methods Enzymol 178: 497-515 (1989); Lamoyi, Methods Enzymol 121: 652-63 (1986); Rousseaux et al., Methods Enzymol 121: 663-9 (1986); Bird and Walker, Trends Biotechnol 9: 132-7 (1991)).
  • An antibody may be modified by conjugation with a variety of molecules, such as polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the present invention provides for such modified antibodies.
  • the modified antibody can be obtained by chemically modifying an antibody. These modification methods are conventional in the field.
  • an antibody of the present invention may be obtained as a chimeric antibody, between a variable region derived from nonhuman antibody and the constant region derived from human antibody, or as a humanized antibody, including the complementarity determining region (CDR) derived from nonhuman antibody, the frame work region (FR) and the constant region derived from human antibody.
  • CDR complementarity determining region
  • FR frame work region
  • Such antibodies can be prepared according to known technology.
  • Humanization can be performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody (see, e.g., Verhoeyen et al., Science 239:1534-1536 (1988)). Accordingly, such humanized antibodies are chimeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • Fully human antibodies including human variable regions in addition to human framework and constant regions can also be used. Such antibodies can be produced using various techniques known in the art. For example, in vitro methods involve use of recombinant libraries of human antibody fragments displayed on bacteriophage (e.g., Hoogenboom & Winter, J. Mol. Biol. 227:381 (1991). Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described, e.g., in U.S. Patent Nos. 6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016.
  • Antibodies obtained as above may be purified to homogeneity.
  • the separation and purification of the antibody can be performed according to the separation and purification methods used for general proteins.
  • the antibody may be separated and isolated by the appropriately selected and combined use of column chromatographies, such as affinity chromatography, filter, ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel electrophoresis and isoelectric focusing (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), but are not limited thereto.
  • a protein A column and protein G column can be used as the affinity column.
  • Exemplary protein A columns to be used include, for example, Hyper D, POROS and Sepharose F.F. (Pharmacia).
  • ELISA enzyme-linked immunosorbent assay
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • the antibody of the present invention is immobilized on a plate, a peptide of the present invention is applied to the plate, and then a sample containing a desired antibody, such as culture supernatant of antibody producing cells or purified antibodies, is applied. Then, a secondary antibody that recognizes the primary antibody and is labeled with an enzyme, such as alkaline phosphatase, is applied, and the plate is incubated.
  • a desired antibody such as culture supernatant of antibody producing cells or purified antibodies
  • an enzyme substrate such as p-nitrophenyl phosphate
  • the absorbance is measured to evaluate the antigen binding activity of the sample.
  • a fragment of the peptide such as a C-terminal or N-terminal fragment, may be used as the antigen to evaluate the binding activity of the antibody.
  • BIAcore Pharmacia
  • the above methods allow for the detection or measurement of a peptide of the present invention, by exposing an antibody of the present invention to a sample presumed to contain a peptide of the present invention, and detecting or measuring the immune complex formed by the antibody and the peptide. Because the method of detection or measurement of the peptide according to the present invention can specifically detect or measure a peptide, the method can find use in a variety of experiments in which the peptide is used.
  • the present invention also provides a vector and host cell into which a nucleotide encoding the peptide of the present invention is introduced.
  • a vector of the present invention may be used to keep a nucleotide, especially a DNA, of the present invention in host cell, to express a peptide of the present invention, or to administer a nucleotide of the present invention for gene therapy.
  • E. coli When E. coli is a host cell and the vector is amplified and produced in a large amount in E. coli (e.g., JM109, DH5 alpha, HB101 or XL1Blue), the vector should have "ori" to be amplified in E. coli and a marker gene for selecting transformed E. coli (e.g., a drug-resistance gene selected by a drug such as ampicillin, tetracycline, kanamycin, chloramphenicol or the like).
  • a marker gene for selecting transformed E. coli e.g., a drug-resistance gene selected by a drug such as ampicillin, tetracycline, kanamycin, chloramphenicol or the like.
  • M13-series vectors, pUC-series vectors, pBR322, pBluescript, pCR-Script, etc. can be used.
  • the vector should have a promoter, for example, lacZ promoter (Ward et al., Nature 341: 544-6 (1989); FASEB J 6: 2422-7 (1992)), araB promoter (Better et al., Science 240: 1041-3 (1988)), T7 promoter or the like, that can efficiently express the desired gene in E. coli.
  • a promoter for example, lacZ promoter (Ward et al., Nature 341: 544-6 (1989); FASEB J 6: 2422-7 (1992)), araB promoter (Better et al., Science 240: 1041-3 (1988)), T7 promoter or the like, that can efficiently express the desired gene in E. coli.
  • the host is preferably BL21 which expresses T7 RNA polymerase
  • the vector may also contain a signal sequence for peptide secretion.
  • An exemplary signal sequence that directs the peptide to be secreted to the periplasm of the E. coli is the pelB signal sequence (Lei et al., J Bacteriol 169: 4379 (1987)).
  • Means for introducing of the vectors into the target host cells include, for example, the calcium chloride method, and the electroporation method.
  • expression vectors derived from mammals for example, pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic Acids Res 18(17): 5322 (1990)
  • pEF for example, "Bac-to-BAC baculovirus expression system” (GIBCO BRL), pBacPAK8)
  • expression vectors derived from plants e.g., pMH1, pMH2
  • expression vectors derived from animal viruses e.g., pHSV, pMV, pAdexLcw
  • expression vectors derived from retroviruses e.g., pZIpneo
  • expression vector derived from yeast e.g., "Pichia Expression Kit” (Invitrogen), pNV11, SP-Q01
  • Bacillus subtilis e.g., pPL608, pKTH50
  • Synthesis of peptides derived from TTK 9-mer and 10-mer peptides derived from TTK were designed based on binding affinity prediction against HLA-A*0201 molecule (SEQ ID NOs: 1 to 38).
  • Peptides were synthesized by SIGMA (Sapporo, Japan) or Biosynthesis Inc. (Lewisville, TX) according to a standard solid phase synthesis method and purified by reversed phase high performance liquid chromatography (HPLC). The purity (>90%) and the identity of the peptides were determined by analytical HPLC and mass spectrometry analysis, respectively.
  • Peptides were dissolved in dimethylsulfoxide (DMSO) at 20 mg/ml and stored at -80 degrees C.
  • DMSO dimethylsulfoxide
  • DCs In vitro CTL Induction Monocyte-derived dendritic cells (DCs) were used as antigen-presenting cells (APCs) to induce cytotoxic T lymphocyte (CTL) responses against peptides presented on human leukocyte antigen (HLA). DCs were generated in vitro as described elsewhere (Nakahara S et al., Cancer Res 2003 Jul 15, 63(14): 4112-8). Specifically, peripheral blood mononuclear cells (PBMCs) isolated from a normal volunteer (HLA-A*0201 positive) by Ficoll-Plaque (Pharmacia) solution were separated by adherence to a plastic tissue culture dish (Becton Dickinson) so as to enrich them as the monocyte fraction.
  • PBMCs peripheral blood mononuclear cells isolated from a normal volunteer (HLA-A*0201 positive) by Ficoll-Plaque (Pharmacia) solution were separated by adherence to a plastic tissue culture dish (Becton Dickinson) so as to enrich them as the mon
  • the monocyte-enriched population was cultured in the presence of 1,000 U/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF) (R&D System) and 1,000 U/ml of interleukin (IL)-4 (R&D System) in AIM-V Medium (Invitrogen) containing 2% heat-inactivated autologous serum (AS). After 7 days of culture, the cytokine-induced DCs were pulsed with 20 micro-g/ml of each of the synthesized peptides in the presence of 3 micro-g/ml of beta 2-microglobulin for 3 hrs at 37 degrees C in AIM-V Medium.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • IL interleukin-4
  • AS heat-inactivated autologous serum
  • the generated cells appeared to express DC-associated molecules, such as CD80, CD83, CD86 and HLA class II, on their cell surfaces (data not shown).
  • DC-associated molecules such as CD80, CD83, CD86 and HLA class II
  • These peptide-pulsed DCs were then inactivated by X-irradiation (20 Gy) and mixed at a 1:20 ratio with autologous CD8+ T cells, obtained by positive selection with CD8 Positive Isolation Kit (Dynal). These cultures were set up in 48-well plates (Corning); each well contained 1.5 x 10 4 peptide-pulsed DCs, 3 x 10 5 CD8+ T cells and 10 ng/ml of IL-7 (R&D System) in 0.5 ml of AIM-V/2% AS medium.
  • CTL Expansion Procedure CTLs were expanded in culture using the method similar to the one described by Riddell et al. (Walter EA et al., N Engl J Med 1995 Oct 19, 333(16): 1038-44; Riddell SR et al., Nat Med 1996 Feb, 2(2): 216-23). A total of 5 x 10 4 CTLs were resuspended in 25 ml of AIM-V/5% AS medium with 2 kinds of human B-lymphoblastoid cell lines, inactivated by Mitomycin C, in the presence of 40 ng/ml of anti-CD3 monoclonal antibody (Pharmingen).
  • CTL activity was tested on the 14th day, and CTL clone was expanded using the same method as described above (Uchida N et al., Clin Cancer Res 2004 Dec 15, 10(24): 8577-86; Suda T et al., Cancer Sci 2006 May, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005 Aug, 96(8): 498-506).
  • the cDNA encoding an open reading frame of target genes or HLA-A*0201 was amplified by PCR.
  • the PCR-amplified products were cloned into a vector.
  • the plasmids were transfected into COS7, which is the target genes and HLA-A*0201-negative cell line, using lipofectamine 2000 (Invitrogen) according to the manufacturer's recommended procedures. After 2days from transfection, the transfected cells were harvested with versene (Invitrogen) and used as the target cells (5 X 10 4 cells/ well) for CTL activity assay.
  • the CTL clone was established by limiting dilution from the CTL lines as described in "Materials and Methods", and IFN-gamma production from the CTL clones against target cells pulsed peptide was determined by IFN-gamma ELISA assay. Potent IFN-gamma productions were determined from the CTL clone stimulated with TTK-A02-9-593 (SEQ ID NO: 3) ( Figure 2g).
  • Specific CTL activity against target cells expressing TTK and HLA-A*0201 The established CTL lines and clones raised against each peptide were examined for the ability to recognize target cells that express TTK and HLA-A*0201 molecule.
  • Specific CTL activity against COS7 cells which transfected with both the full length of TTK and HLA-A*0201 gene was tested using the CTL lines and clones raised by corresponding peptide as the effecter cells.
  • COS7 cells transfected with either full length of TTK genes or HLA-A* 0201 were prepared as controls.
  • the CTL line and the CTL clone stimulated with TTK-A02-9-593 showed potent CTL activity against COS7 cells expressing both TTK and HLA- A* 0201 (a: line, b: clone).
  • the CTL clone established with TTK-A02-9-593 also showed specific CTL activity against a tumor cell line endogenously expressing both TTK and HLA- A* 0201: H1650 cells (c).
  • no significant specific CTL activity was detected against controls: TE1 cells and PC3 cells.
  • TTK-A02-9-593 SEQ ID NO: 3
  • TTK-A02-9-593 SEQ ID NO: 3
  • TTK-A02-9-593 The CTLs stimulated with TTK-A02-9-593 (SEQ ID NO: 3) showed significant and specific CTL activity. This result may be due to the fact that the sequences of TTK-A02-9-593 (SEQ ID NO: 3) are homologous to peptides derived from other molecules that are known to sensitize the human immune system. To exclude this possibility, homology analyses were performed for these peptide sequences using as queries the BLAST algorithm (www.ncbi.nlm.nih.gov/blast/blast.cgi) which revealed no sequence with significant homology. The results of homology analyses indicate that the sequences of TTK-A02-9-593 (SEQ ID NO: 3) are unique and thus, there is little possibility, to our best knowledge, that these molecules raise unintended immunologic response to some unrelated molecule.
  • TTK-A2-9-462 SEQ ID NO: 1
  • TTK-A02-9-547 SEQ ID NO: 5
  • TTK-A2-9-719 SEQ ID NO: 6
  • TTK-A2-10-462 SEQ ID NO: 22
  • TTK-A02-9-593 (SEQ ID NO: 3) identified in the present invention possesses a distinctly effective activity that increases by one or two orders of magnitude in the IFN-gamma production ( Figure 3). Therefore, the peptide of the present invention appears to be a promising target of the treatment, prophylaxis, and/or prevention of cancer, including, but not limited to, lung cancer, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophageal cancer, gastric cancer, diffused type gastric cancer, liver cancer, NSCLC, lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal carcinoma, SCLC, soft tissue tumor and testicular tumor.
  • cancer including, but not limited to, lung cancer, bladder cancer, breast cancer, cervical cancer, cholangiocellular carcinoma, CML, colorectal cancer, endometriosis, esophageal cancer, gastric cancer, diffused type gastric

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JP7274223B2 (ja) * 2017-07-12 2023-05-16 ノイスコム アーゲー マイクロサテライト不安定性（ｍｓｉ）癌の予防及び治療のための共通腫瘍ネオアンチゲンをベースとした万能ワクチン
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